1. Field of the Invention
Embodiments of the invention relate to a bacterial high-expression system and its use for screening of recombinant clones from single chain (sc) antibody libraries and simultaneous isolation of the desired ScFv based upon the screening. Also disclosed are scFv antibodies to interferon α2b produced by the method.
2. Description of the Related Art
1. Single Chain Variable Fragment (scFv) Antibody
Antibodies hold a firm place in biological research and have an increasingly important role in medical and industrial applications. Antibodies are highly selective binding agents and can be generated against any substance by standard approaches. In particular, monoclonal antibodies provide homogenous antibodies of predefined specificity. Antibody fragments can be generated in E. coli that have the same affinity as the complete antibody. Methods for the generation of large repertoires of diverse antibody molecules in bacteria has been described (Hurse, et al. (1989) Science 246: 1275-1281).
The smallest portion containing an antigen-binding site is the variable fragments (Fv) of an antibody. There are variable fragments on both the light and heavy chains. In single chain Fv (scFv) the two antigen binding variable regions of the light and heavy chain (VH Fv and VL Fv) are artificially connected by a linker peptide, designated as single chain variable fragment or single chain antibody (Bird, et al. (1988) Science 242:423-426; Orlandi, et al (1989) Proc Natl Acad Sci USA 86:3833-3837; Clarkson et al., Nature 352: 624-628 (1991)). The antigen binding site is made up of the variable domains of light and heavy chains of a monoclonal antibody. Several investigations have shown that the Fv fragment has indeed the full intrinsic antigen binding affinity of one binding site of the whole antibody. To stabilize the association of the recombinant Fv fragments, the fragments were joined with a short peptide linker and expressed as a single polypeptide chain. A variety of linker peptides, generally of length 12-25 aa, were tested and did not disturb the proper folding of the VH and VL domains (Bird, et al. (1988) ibid; Huston et al (1988) Proc Natl Acad Sci: USA 85: 5879-5883).
A frequently used linker for scFv antibodies is (Gly4Ser)3, a single 15 amino acid peptide with 12 glycines and 3 serines that bridges the ˜4.5 nm (theoretical distance 5.4 nm, Hudson P (1995) Structure and application of single-chain Fvs as diagnostic and therapeutic agents. In: H. Zola (ed), Monoclonal antibodies the second generation. BIOS Scientific Publishers Limited, Oxford, UK.) gap between the C terminus of one domain and the N terminus of the other and has a flexible structure with inhanced mobility (Huston, et al., (1988) ibid; Freund, et al (1993) FEBS Lett 320:97-100). This construction facilitates chain pairing and minimizes refoldings and aggregations encountered when the two chains are expressed individually.
ScFv antibodies have the following advantages:    1. ScFv antibodies overcome the problem of dissociation of VH and VL often encountered with Fv fragments.    2. ScFv antibodies provide immunologically active molecules of conveniently small size.    3. ScFv antibodies can be produced on a large scale by fermentation with high purity and at low cost.    4. ScFv antibodies can be easily genetically manipulated.
ScFv antibodies is a broad term and is used herein in its usual sense. In particular, the term ScFv includes scFv antibodies, recombinant phage display of scFv antibodies, dimeric forms of scFv antibodies, dimeric forms of scFv or miniantibodies, bi-specific scFv (diabodies) and multimeric ScFv forms.
Small scFv fragments are considered promising for medical and biological applications because of superior tissue penetration, absence of side reactions involving the constant domains, as well as engineering of fusion protins, such as scFv-coupled toxins, the creation of multivalent or bispecific proteins or Antibody directed enzyme prodrug therapy (ADEPT) (Syrigos, et al (1999) Anticancer Res 19:605-613).
2. Selection Strategies
The use of currently developed techniques such as phage display (Smith G P (1985) Science 228:1315-1317; Winter, et al. (1994) Annu Rev Immunol 12:433-455; Dunn I S (1996) Curr Opin Biotechnol 7:547-553), ribosome display (Mattheakis, et al. (1994) Proc Natl Acad Sci USA 91:9022-9026; Hanes, et al. (1997) Proc Natl Acad Sci USA 94:4937-4942), yeast surface display (Bader E T, et al. (1997) Nature Biotechnol 15:553-557) and bacterial display (Daugherty, et al. (1998) Protein Eng 11:825-832) for in vitro selection of molecular interactions under evolutionary pressure has provided a new perspective in antibody engineering. Phage display has been most widely used (Cortese, et al. (1996) Curr Opin Biotechnol 7:616-621; Hoogenboom, et al. (1998) Immunotechnology 4:1-20). Phage display relies on fusing the protein of interest to a minor coat protein of the phage, the gene3 protein (g3p). In phage display, a ligand (e.g. an antigen) is immobilized and a collection of binding proteins (e.g. antibodies) are displayed on the phage, that is, provided as a fusion with the g3p (McCafferty et al., Nature, 348: 552-554 (1990); Soderlind et al., Immunol. Reviews 130: 109-124 (1992); Winter, et al. (1994) Annu Rev Immunol 12:433-455). The general technique for filanentous phage display is described in U.S. Pat. No. 5,658,727. The essential trick is that the genetic information of the displayed protein is contained within the phage DNA in the same phage particle and thus, physically connected to the expressed protein.
Antibody phage display requires that a repertoire or library of immunoglobulin-encoding genes be cloned into the filamentous phage. The library is accomplished by amplifying the variable region of immunoglobulin fragments or germline V-genes. The PCR products are cloned into a filamentous phage to incorporate a heavy chain and a light chain variable region cDNA copy connected by a linker and expressed on the surface of the filamentous phages. The phages without binding ability will be removed by washing. The remaining phages are used to infect E. coli for their amplification. The selection procedure, the so-called panning, can be repeated with increasing stringency to select clones with the highest affinity (Mersmann, et al. (1998) J Immunol Methods 220:51-58). Panning (as described by Parmley, et al. Gene 73: 305-318 (1988)) and is preferred because high titers of phage can be screened easily, quickly and in small volumes. Furthermore, this procedure can select minor antibody fragment species within the population which otherwise would have been undetectable and amplify them to achieve a substantially homogenous population.
Phage display technology has been applied in many fields within the biological and medical sciences for study of molecular interactions and especially in the generation of monoclonal antibodies. However, high costs and time-consuming processes involving several rounds of panning and phage rescue are an intrinsic problem of a phagemid-based display system. The disadvantage of this approach also is that the yield of antibodies obtained using secretion vectors is relatively low. In most cases it is possible to avoid several cycles of phage rescue after antigen-affinity selection procedures which simplifies detection when screening a large numbers of clones. Other methods for producing divers libraries of antibodies and screening for desirable binding specificities are described in U.S. Pat. Nos. 5,667,988 and 5,759,817. The enriched antibodies are also screened with additional detection techniques such as expression colony lift (Young, et al. (1983) Science 222: 778-782, incorporated herein by reference) or cell surface display (U.S. Pat. No. 5,866,344). Vectors for this purposes are described in U.S. Pat. No. 5,348,867. Such methods are applicable for detection in situ of colonies expressing recombinant antibodies having the desired characteristics. Embodiments of the present invention are directed to the development of a bacterial expression system for simultaneous screening of large numbers of recombinant clones from preliminary selected antibody libraries. But in contrast to published protocols, after screening, the clones, which express the recombinant antibodies to desired antigen, can be directly used for large-scale production of the ScFvs.
3. Expression Strategies for ScFv in Escherichia coli 
a. Expression by Secretion
Embodiments of the invention address the need to find protein expression systems that are convenient and highly-productive for large-scale production. There are several ways to express antibody fragments in E. coli and there are some advantages common to all various approaches. But there is no single expression strategy for antibody fragments now. The choice depends very critically on the intended application, be it the mass production of a single antibody species, the rapid engineering of an antibody, its structure determination, the testing of many variants or the screening of libraries. Requirements will also differ for antibodies intended for human or animal use as opposed to those intended for in vitro research or industrial purposes only.
Many strategies and vector constructions have been used for the expression of antibody fragments in E. coli. One way to obtain ScFv in a biologically active form is functional expression by secretion. The secretion of the ScFv gives rise to native and functional antibody fragments, and leads to many of the attractive features of a bacterial expression system, notably the screening of binding activity without prior in vitro folding. The essence of the strategy is to reproduce in E. coli the normal folding and assembly pathway of antibodies within the eukaryotic cell. In antibody producing cells, the two chains are expressed separately as precursors with N-terminal signal sequences and separately transported to the lumen of the endoplasmic reticulum (ER). There, the signal sequences are cleaved by a membrane-bound signal peptidase. In the lumen of the ER, folding of the protein, disulfide bond formation and assembly of the complete antibody take place.
The main hypothesis in the design of the secretory expression system for antibody fragments was that protein transport to the periplasm of E. coli is functionally equivalent to the transport of a protein to the lumen of the ER. A system was designed that directs ScFv to the periplasm of the same E. coli cell. The main advantage of this secretory expression system is that it directly leads to an assembled functional product with correctly formed disulfide bonds without the need to refold the protein in vitro. Generally, the desired antibody fragment is fused to an amino acid sequence that includes the signals for localization to the outer membrane and for translocation across the outer membrane. The amino acid sequences responsible for localization and for translocation across the outer membrane may be derived either from the same bacterial protein or from different proteins of the same or different bacterial species or from some bacteriophages. A wide variety of signal peptides have been used successfully in E. coli for protein translocation to the periplasm. These include prokaryotic signal sequences, such as the E. coli signals PhoA (Denefle, et al. (1989) Gene 85:499-510), OmpA (Denefle et al., 1989, ibid; Ghrayeb, et al. (1984) EMBO J. 3:2437-2442; Goldstein, et al. (1990) J. Bacteriol. 172:1225-1231), OmpT (Johnson, et al. (1996) Protein Expression Purif. 7:104-113), LamB and OmpF (Hoffman, et al. (1985) Proc. Natl. Acad. Sci. USA 82:5107-5111), b-lactamase (Kadonaga, et al. (1984) J. Biol. Chem. 259:2149-2154), Pe1B from Erwinia carotovora (Better, et al. (1988) Science 240: 1041-1043; Lei, et al. (1987) J. Bacteriol. 169: 4379-4383), leader sequences cpVIII and cpVIII from M13 filamentous phage coat proteins. The disadvantage of this approach is that protein yield is relatively low in most cases reported (Skerra, A. (1993) Current Opinion in Immunology 5, 256-262; Raag, et al. (1995) FASEB Journal 9, 73-80), and this places certain limitations on the use of such systems for preparative obtaining of recombinant-antibodies. The present invention represents the development a bacteria high-expression system which can be used for large scale production of the ScFvs as inclusion bodies.
b. Cytoplasmic Expression
The second approach is to produce scFv or Fv as insoluble cytoplasmic inclusion bodies. This strategy was used in the first reports on expressing antibodies in E. coli (Boss et al (1984) Nucleus Acid Res 12: 3791-3806; Cabilly et al. (1984) Proc Natl Acad Sci USA 81: 3273-3277). All types of antibody fragments (Fab, Fv, ScFv) have been produced in this way (Bird, et al. (1988) Science 242:423-426; Huston et al (1988) Proc Natl Acad Sci: USA 85: 5879-5883; Field, et al. (1990) Protein Eng. 3: 641-647; Pantoliano et al (1991) Biochemistry 30: 10117-10125; Cheadle et al (1992). Mol Immunol 29: 21-30) and a variety of strains, plasmids and promoters have been used. The T7 system, as a particularly strong, but regulatable system, was found useful (Huston et al (1991) Methods Enzymol 203: 46-88, Freund, et al. (1993) FEBS Lett 320:97-100). For many years, expression systems which produced soluble secreted recombinant proteins were favored over systems which produced ScFv as inclusion bodies because of the difficulties encountered when refolding inclusion body proteins; however, careful examination of the folding conditions allowed researchers to find ways to refold disulphide bonded proteins with relatively high yields. The development of improved methods for refolding ScFvs would greatly enhance their availability and utility.
However, since inclusion bodies contain mis-folded proteins that lack biological activity, the expression of antibodies of interest cannot be monitored directly by functional assay. Embodiments of the present invention relate to development of a bacterial high-expression system which is useful for screening antibody libraries by direct functional assay. Linkage of overexpression and screening in the developed system is accomplished by the combination of targeting some ScFvs into the periplasm of bacterial cell to allow for convenient screening of the library member of interest and the formation of inclusion bodies in the cytoplasm. Periplasmic targeting is provided by the presence of a secretory leader peptide at the N-terminus. Formation of cytoplasmic inclusion bodies is provided by the presence of the strong promoter T7 in the expression vector.
Expression of single chain antibody fragments as inclusion bodies is advantageous due to the very high levels of enriched protein produced and the protection of the protein product from proteolytic degradation. In addition, when producing a recombinant product which can be toxic or lethal to the host cell, the inclusion body protects the host from toxic and/or lethal effects. ScFv proteins produced in inclusion bodies have been successfully refolded. Properly folded proteins can be produced from inclusion bodies using a variety of solubilization and refolding schemes (Rudolph, et al. (1996). FASEB J. 10, 49-56; Marston, et al. (1990). Methods Enzymol. 182, 264-276 which are incorporated herein by reference). But the key to a successful commercial refolding process lies in achieving high yields while refolding at high protein concentrations (De Bemardez Clark, E. (1998). Curr. Opin. Biotechnol. 9, 157-163). Embodiments of the present invention also include optimization of the refolding process for ScFv to human interferon a2b. The developed method is applicable for large-scale production of biologically active ScFv hexahistidine proteins in bacterial inclusion bodies.